Antibodies that bind to an epitope on the huntington&#39;s disease protein

ABSTRACT

The present invention relates generally to the generation and characterization of anti-huntingtin antibodies binding an epitope on the Huntington&#39;s disease protein. The invention further relates to the use of such anti-huntingtin antibodies in the diagnosis and treatment of Huntington&#39;s disease.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/354,246, which in turn claims priority under 35 U.S.C.§119(e) from U.S. provisional application No. 60/353,032, filed on Jan.28, 2002. All of the priority applications are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to antibodies to Huntington'sdisease protein as well as methods and means for making and using suchantibodies.

2. Description of the Related Art

Huntington's disease (HD) is a fatal autosomal dominantneurodegenerative disorder that is caused by the extension of apolyglutamine (polyQ) tract in exon 1 the protein huntingtin (Htt) to alength of greater than 40 units (Reddy et al. Trends Neurosci.22:248-255 (1999)). The huntingtin gene is known and the subject of U.S.Pat. No. 5,693,757. Mutant Htt with greater than 40 CAG repeats gains atoxic function and induces death in subpopulations of neurons in thestriatum and cortex (Zoghbi et al. Annu. Rev. Neurosci. 23:217-247(2000); Tobin et al. Trends Cell Biol. 10:531-536 (2000)). Neuronaldeath in HD has been attributed not only to polyQ toxicity, but also toactivation of caspases, interference with transcriptional machinery, andsequestration/inactivation of wild-type Htt and other important cellularfactors.

A hallmark of HD and other polyQ diseases is the formation of insolubleprotein aggregates in affected neurons (Ross Neuron 19:1147-1150 (1997);Wanker Biol. Chem. 937-942 (2000). Immunohistochemistry and subcellularfractionation indicate that Htt is normally located in the cytoplasmwhile the mutant form of Htt is also found in aggregates in the nucleus(Ferrigno et al. Neuron 26:9-12 (2000)). A major component of theaggregates in HD is the N terminus exon 1 of mutant Htt. As normalhuntingtin protein is localized in the cytoplasm and mutant huntingtinprotein is found in aggregates, also known as and referred to asinclusions, in the nucleus (Ferrigno et al., Neuron, 26:9-12 (2000)),translocation of mutant huntingtin protein to the nucleus is believed tobe important in the pathogenesis of HD.

Because there is no current treatment available for this disease, thereis a clear need for new treatments for Huntington's disease. Moleculesthat block the toxic effects of Htt itself or the lethal consequences ofits binding to other proteins have good potential for therapeutic use.Thus, antibodies may serve as treatments for Huntington's disease. Anantibody termed 1C2 is described in WO 97/17445. Finkbeiner (U.S. Pat.No. 6,291,652) provides antibodies specific for proteins havingpolyglutamine expansions. In particular, Finkbeiner provides antibodieshaving a higher affinity than an antibody identified as 1C2.

SUMMARY OF THE INVENTION

In one aspect, the invention involves antibodies, specificallymonoclonal antibodies including antibody fragments, such as single-chainvariant fragments, and mimetics thereof (including intrabodies), to thehuntingtin protein. Preferred biological activities of the antibodiesinclude the capability of preventing cell death or apoptosis, preventingmutant huntingtin protein aggregation and the regulating the toxiceffects of mutant huntingtin protein that are associated withneurodegenerative disease. In one embodiment, the antibodies bindspecifically to an epitope within a polyproline region of the huntingtinprotein comprising greater than 5 consecutive proline residues and arecapable of inhibiting aggregation of huntingtin protein. In anotherembodiment, the antibodies bind specifically to an epitope within thepolyglutamine region of the huntingtin protein comprising greater than 6consecutive glutamine residues and are capable of stimulatingaggregation of huntingtin protein. In another embodiment, the antibodiesspecifically interact with an amino acid epitope within the carboxyterminus of the protein encoded by exon 1 of the huntingtin protein,said carboxy terminus comprising the amino acid sequence of SEQ ID NO:2. In another embodiment, the antibodies are in association with atherapeutically acceptable carrier. The single-chain variant antibodyfragments are encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 3, 4, 5 and 6.

The methods of the invention involve the treatment of an individual,preferably a patient, more preferably a mammalian patient and even morepreferably a human mammalian patient, having or suspected of havingHuntington's disease by administering a therapeutically effective amountof an antibody, such as a single-chain variant fragment, or antibodycomposition comprising a single-chain variant fragment to theindividual. The antibody compositions of the methods are preferablydelivered intracranially, for example, by injection directly into braintissue or by injection into the cerebrospinal fluid.

The methods of the invention may also involve the treatment ofHuntington's disease by expressing anti-huntingtin antibodies, includingsingle-chain variant fragments, in cells expressing mutant huntingtinprotein. Nucleic acids encoding the subject antibodies and methods fortheir expression, including in therapeutic treatment protocols, areprovided. Nucleic acids of the invention can be introduced into a hostcell using various viral vectors and non-viral delivery techniques forexpression of the nucleic acid encoding the antibody in brain tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows epitope mapping of anti-huntingtin antibodies MW1-MW8 bypeptide array which includes both human and mouse huntingtin peptides.Two rows of peptide dot blots are shown for each of the MW1-MW8anti-huntingtin antibodies with the upper row corresponding to thepeptides shown at the top of the figure, and the lower row correspondingto the peptides shown at the bottom of the figure. The three types ofepitope of the huntingtin protein are underlined in the correspondingpeptide sequences ______=polyQ; ...=polyP; _(———)=C terminus).

FIG. 2 shows a diagram of the epitope mapping results from the peptidearray analysis in FIG. 1. The results of the peptide array are displayedon a linear diagram of the normal human huntingtin amino acid sequence(SEQ ID NO: 1).

FIG. 3 shows a Western blot of normal (WT) and transgenic 94Q knock-in(94Q) mouse cerebellum extracts using anti-huntingtin antibodiesMW1-MW8. Control antibodies 1C2 and 1F8 were used to identify mutanthuntingtin protein, and 2166 antibody was used to identify both mutantand normal huntingtin protein.

FIG. 4 shows a Western blot of normal (HD7) and Huntington's disease(HD2) human lymphoblastoma cell extracts using anti-huntingtinantibodies MW1-MW8. Control antibodies 1C2 and 1F8 were used to identifymutant huntingtin protein, and 2166 antibody was used to identify bothmutant and normal huntingtin protein.

FIGS. 5A-5E show immunofluorescence staining patterns of MW1anti-huntingtin and control 1C2 antibodies in wild-type (WT) and R6/2transgenic cortex (R6), having mutant spinal cord neurons. FIG. 5A showsthe level of background immunostaining in the absence of primaryantibody. FIG. 5B show immunostaining of MW1 and 1C2 antibodies incortical neurons. FIGS. 5D and 5E shows immunostaining of MW1 and 1C2antibodies in fresh frozen R6/1 cortex sections, respectively.

FIGS. 6A-6H show immunofluorescence staining patterns of MW2-MW5anti-huntingtin and control 1F8 antibodies in wild-type (WT) and R6/2transgenic cortex (R6), having mutant spinal cord neurons. MW2 (FIG.6A), MW3 (FIG. 6B), MW4 (FIG. 6C), MW5 (FIG. 6D) and control 1F8 (FIG.6E) antibodies exhibit similar patterns, neuronal Golgi complexstaining, when used to stain spinal cord sections. MW3 staining ofparaformaldehyde fixed spinal cord sections from R6/2 mice is shown inFIG. 6F. MW3 staining of wild-type and R6/2 mutant brain sections areshown in FIGS. 6G and 6H, respectively.

FIGS. 7A-7I show immunofluorescence staining patterns of MW6-MW8anti-huntingtin antibodies in wild-type (WT) and mutant transgenic R6/2(R6) spinal cord and brain. FIGS. 7E-7H show a confocal series of MW7staining. MW6 shows punctate staining of the neuropil in WT (FIG. 7A)and R6/2 spinal cord while MW7 shows punctate staining of theperinuclear or nuclear membrane in WT (FIG. 7C) and R6/2 (FIG. 7D)brain. MW8 shows staining of neuronal inclusions in R6/2 (8-week old)fixed cortex sections (FIG. 7J).

FIG. 8 shows a diagram illustrating the binding patterns of the MW1-MW8anti-huntingtin antibodies to the huntingtin protein as analyzed bypeptide array and immunohistochemical staining in vivo. The domainstructure of the diagram of the huntingtin protein is from left to rightas follows: the N-terminus, the polyQ domain, the polyP domain and theC-terminus.

FIG. 9 shows coimmunoprecipitation of expressed MW scFv proteins fromlysates of 293 cells cotransfected with Htt exon 1-EGFP, either25-residue polyQ (PQ25) or 103-residue polyQ (PQ103), and a Flag-scFv orFlag-IκBα.

FIG. 10 shows expression of hMW9 scFv and a control scFv (C) as analyzedby in vitro transcription and translation of hMW9 scFv in the presenceof ³⁵S-methionine. The scFv was incubated with 5 μg of recombinantGST-HDx-1 bound to gluthathione beads and subsequent analysis of thescFv that bound to the glutathione beads by SDS-PAGE andautoradiography.

FIG. 11 shows immunofluorescence staining of 293 cells transfected withMW1, MW2 and MW7 scFvs or a control empty scFv vector (C) with anti-Flagantibodies two days after transfection and subsequent fixation.

FIG. 12 shows the effects of the expression of hMW9 scFv, empty plasmid(C) or control plasmid (cscFv) and HDx-1, containing 103 polyQ and fusedto GFP in human 293 cells as analyzed by fluorescence microscopy.

FIG. 13 shows colocalization of MW1, MW2 or MW7 scFv with mutant Htt in293 cells cotransfected with mutant Htt fused to EGFP tag and scFvtagged with a Flag tag.

FIG. 14 shows colocalization of MW8 scFv with mutant Htt in 293 cellscotransfected with mutant Htt fused to EGFP tag and scFv tagged with aFlag tag.

FIG. 15 shows inhibition of Htt-induced cell death in 293 cells with MW7scFv and enhancement of Htt-induced cell death in 293 cells with MW1 orMW2 scFvs. 293 cells were transfected with Htt exon 1-EGFP and an emptyvector (control) or one of the anti-huntingtin scFvs, MW1, MW2 or MW7tagged with a Flag tag. The transfected cells were visualized by GFPfluorescence, and dying cells by TUNEL staining. The presence of MW7scFv decreases the number of TUNEL+ cells.

FIG. 16 shows inhibition of Htt-induced cell death in 293 cells with MW8scFv. 293 cells were transfected with Htt exon 1-EGFP and an emptyvector (control) or MW8 anti-huntingtin scFv, tagged with a Flag tag.The transfected cells were visualized by GFP fluorescence, and dyingcells by TUNEL staining. The presence of MW8 scFv decreases the numberof TUNEL+ cells.

FIG. 17 shows a chart representing quantitation of the effects of theexpression of anti-huntingtin antibodies, MW1, MW2 and MW7 on mutant Htttoxicity. MW1 and MW2 exacerbated Htt-induced cell death while MW7inhibited Htt toxicity.

FIGS. 18A-18B show reduction of aggregation of mutant huntingtin proteinin 293 cells as analyzed by Western blotting. Lysates of 293 cellstransfected with mutant Htt and an scFv, MW1, MW2 or MW7, were subjectedto high-speed centrifugation and were analyzed by Western blotting withanti-HD₁₋₁₇ antibodies. The Htt in the pellet that can be solubilized bySDS treatment is about 80 kDa, whereas the Htt that cannot besolubilized does not enter the gel and is visualized as a band at thetop of the gel (FIG. 18A). FIG. 18B shows that the level of soluble Httin the cleared lysates does not appear to be affected by expression ofMW1, MW2 or MW7 scFv expression.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The huntingtin protein comprises a number of distinct regions that arebelieved to play a role in the toxicity of mutant Htt, as well asinteraction of the Htt protein with other molecules. The presentinvention is based, in part, on the identification of antibodiesdirected to one or more distinct regions of the Htt protein that havedesirable biological activities (Khoshnan et al. Proc. Natl. Acad. Sci.USA 99:1002-1007 (2002); Ko et al. Brain Res. Bull. 56:319-329 (2001),both of which are expressly incorporated herein by reference).

Antibodies, as well as other binding agents, including binding fragmentsand mimetics thereof (including intrabodies), that specifically bind tothe Htt protein are provided. Preferred antibodies specifically bind tothe polyglutamine (“polyQ”) domain polyproline (“polyP”) domain orcarboxy terminus of the huntingtin (Htt) protein.

Nucleic acid sequences encoding the subject antibodies, as well asmethods for their expression, including in therapeutic treatmentprotocols, are also provided.

The preferred binding agents, e.g. antibodies, fragments and mimeticsthereof, etc., bind to the huntingtin protein in a manner that differsin at least one aspect from the 1C2 antibody (Trottier et al., Nature,10:104-110 (1995); Trottier et al., Nature, 378:403-406 (1995)). Forexample, and without limitation, the preferred antibodies may differfrom the 1C2 antibody in terms of the epitope that they recognize or oneor more of specificity, affinity and avidity.

Also provided are methods of screening compounds for the ability tomodulate the activity of proteins comprising a polyglutamine repeat,particularly the huntingtin protein, as well as pharmaceuticalcompositions comprising such agents.

In addition, methods and devices are provided for screening samples forthe presence of proteins comprising a polyglutamine repeat. In aparticularly preferred embodiment, methods for identifying the presenceof mutant huntingtin protein are provided. The methods may be used, forexample, to diagnose a patient as someone who is, or is likely to sufferfrom Huntington's disease or a related disorder.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present invention, the following terms aredefined below.

“Huntingtin” and “Htt” refer broadly to the huntingtin gene and theprotein encoded by the huntingtin gene, including mutant and variantforms as well as native forms. “Variants” are biologically activepolypeptides having an amino acid sequence which differs from thesequence of a native sequence polypeptide. Native sequence humanhuntingtin protein is described, for example, by The Huntington'sDisease Collaborative Research Group in Cell 72:971-983 (1993) as wellas in Li et al. Nature 378:398-402 (1995) and WO 02/29408. The number ofpolyglutamine repeats in native huntingtin protein is known to vary,from about 13 to about 36 glutamine residues in the polyQ region ofnative human protein. Native sequence murine Htt is described, forexample, in Lin et al. Hum. Mol. Genet. 3 (1), 85-92 (1994) andtypically comprises about 7 glutamine residues in the polyQ region.Particular variants of the huntingtin gene will comprise differentnumbers of CAG repeats, resulting in variation in the polyglutamineregion of the huntingtin protein.

“Mutant huntingtin protein” refers to huntingtin protein which differsin some respect from the native sequence huntingtin protein. Typically,mutant huntingtin will comprise an expanded polyglutamine or polyprolineregion compared to the native form. A preferred mutant huntingtinprotein has an expanded polyglutamine region of 40 or more glutamineresidues.

As used herein, “nucleic acid” is defined as RNA or DNA that encodes aprotein or peptide of the invention, particularly an antibody to thehuntingtin protein, is complementary to a nucleic acid sequence encodingsuch peptides, hybridizes to such a nucleic acid and remains stablybound to it under appropriate stringency conditions, exhibits at leastabout 50%, 60%, 70%, 75%, 85%, 90% or 95% nucleotide sequence identityacross the open reading frame, or encodes a polypeptide sharing at leastabout 50%, 60%, 70% or 75% sequence identity, preferably at least about80%, and more preferably at least about 85%, and even more preferably atleast about 90 or 95% or more identity with the peptide sequences.Specifically contemplated are genomic DNA, cDNA, mRNA and antisensemolecules, as well as nucleic acids based on alternative backbones orincluding alternative bases whether derived from natural sources orsynthesized.

As used herein, the terms nucleic acid, polynucleotide and nucleotideare interchangeable and refer to any nucleic acid, whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sultone linkages, andcombinations of such linkages. The terms nucleic acid, polynucleotideand nucleotide also specifically include nucleic acids composed of basesother than the five biologically occurring bases (adenine, guanine,thymine, cytosine and uracil).

The terms “replicable expression vector” and “expression vector” referto a piece of DNA, usually double-stranded, which may have inserted intoit a piece of foreign DNA. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell. The vector is used totransport the foreign or heterologous DNA into a suitable host cell.Once in the host cell, the vector can replicate independently of thehost chromosomal DNA, and several copies of the vector and its inserted(foreign) DNA may be generated. In addition, the vector contains thenecessary elements that permit translating the foreign DNA into apolypeptide. Many molecules of the polypeptide encoded by the foreignDNA can thus be rapidly synthesized.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, and possibly, other as yet poorly understood sequences.Eukaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancer.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or a secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

“Biological property” or “biological activity” is a biological functioncaused by an antibody or other compound of the invention. With regard tothe anti-huntingtin protein antibodies, biological activity refers, inpart, to the ability to specifically bind to the huntingtin protein.Other preferred biological activities include prevention of cell deathor apoptosis, prevention of mutant huntingtin aggregation and theability to regulate the toxic effects of mutant huntingtin protein thatare associated with neurodegenerative disease.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Antigen” when used herein refers to a substance, such as a particularpeptide or protein, that can bind to a specific antibody. Preferredantigens include huntingtin protein, mutant huntingtin protein, andfragments thereof.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins, composed of two identical light (L)chains and two identical heavy (H) chains. Each light chain is linked toa heavy chain by one covalent disulfide bond. while The number ofdisulfide linkages varies among the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intra-chain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end. The constant domain of the light chain isaligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains.

The term “antibody” herein is used in the broadest sense andspecifically covers human, non-human (e.g. murine) and humanizedmonoclonal antibodies, including full length monoclonal antibodies,polyclonal antibodies, multi-specific antibodies (e.g., bispecificantibodies), and antibody fragments, including intrabodies, so long asthey exhibit the desired biological activity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and terminal or internal amino acid sequence by use ofa spinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

“Antibody fragments” comprise a portion of a full-length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; intrabodies; linear antibodies; single-chain antibodymolecules; and multi-specific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of antibodies wherein the individualantibodies comprising the population are identical except for possiblenaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific and are directed against asingle antigenic site. In addition, monoclonal antibodies may be made byany method known in the art. For example, the monoclonal antibodies tobe used in accordance with the present invention may be made by thehybridoma method first described by Kohler et al., Nature 256:495(1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat.No. 4,816,567). The “monoclonal antibodies” may also be isolated fromphage antibody libraries using the techniques described in Clackson etal., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.222:581-597 (1991), for example. The monoclonal antibodies hereinspecifically include antibody fragments, such as single-chain Fv or scFvantibody fragments.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass. Fragments of chimeric antibodies are also includedprovided they exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855(1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are antibodiesthat contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies are generally human immunoglobulins in whichhypervariable region residues are replaced by hypervariable regionresidues from a non-human species such as mouse, rat, rabbit ornon-human primate having the desired specificity, affinity, andcapacity. Framework region (FR) residues of the human immunoglobulin maybe replaced by corresponding non-human residues. In addition, humanizedantibodies may comprise residues that are not found in either therecipient antibody or in the donor antibody. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of thehypervariable regions correspond to those of a non-human immunoglobulinand all or substantially all of the FRs are those of a humanimmunoglobulin sequence. The humanized antibody optionally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

“Single-chain Fv” or “scFv” antibody fragments typically comprise theV^(H) and V_(L) domains of a monoclonal antibody, wherein these domainsare present in a single polypeptide chain. Generally, the Fv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains which enables the scFv to form the desired structure for antigenbinding. For a review of scFv see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994).

The term “epitope” is used to refer to binding sites for (monoclonal orpolyclonal) antibodies on protein antigens. There are many methods knownin the art for mapping and characterizing the location of epitopes onproteins, including solving the crystal structure of an antibody-antigencomplex, competition assays, gene fragment expression assays, andsynthetic peptide-based assays, as described, for example, in Chapter 11of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. A competitionELISA assay is specifically described in Example 1. According to thegene fragment expression assays, the open reading frame encoding theprotein is fragmented either randomly or by specific geneticconstructions and the reactivity of the expressed fragments of theprotein with the antibody to be tested is determined. The gene fragmentsmay, for example, be produced by PCR and then transcribed and translatedinto protein in vitro, in the presence of radioactive amino acids. Thebinding of the antibody to the radioactively labeled protein fragmentsis then determined by immunoprecipitation and gel electrophoresis.Certain epitopes can also be identified by using large libraries ofrandom peptide sequences displayed on the surface of phage particles(phage libraries). Alternatively, a defined library of overlappingpeptide fragments can be tested for binding to the test antibody insimple binding assays. The latter approach is suitable to define linearepitopes of about 5 to 15 amino acids.

An antibody binds “essentially the same epitope” as a referenceantibody, when the two antibodies recognize identical or stericallyoverlapping epitopes. The most widely used and rapid methods fordetermining whether two epitopes bind to identical or stericallyoverlapping epitopes are competition assays, which can be configured inall number of different formats, using either labeled antigen or labeledantibody. Usually, the antigen is immobilized on a 96-well plate, andthe ability of unlabeled antibodies to block the binding of labeledantibodies is measured using radioactive or enzyme labels. A competitionELISA assay is disclosed in Example 1.

The term amino acid or amino acid residue, as used herein, refers tonaturally occurring L amino acids or to D amino acids as describedfurther below with respect to variants. The commonly used one- andthree-letter abbreviations for amino acids are used herein (BruceAlberts et al., Molecular Biology of the Cell, Garland Publishing, Inc.,New York (3d ed. 1994)).

Hybridization is preferably performed under “stringent conditions” whichmeans (1) employing low ionic strength and high temperature for washing,for example, 0.015 sodium chloride/0.0015 M sodium citrate/0.1% sodiumdodecyl sulfate at 50° C., or (2) employing during hybridization adenaturing agent, such as formamide, for example, 50% (vol/vol)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C. Another example is useof 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6/8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1%SDS.

As used herein, “treatment” is a clinical intervention made in responseto a disease, disorder or physiological condition manifested by apatient, particularly Huntington's disease. The aim of treatmentincludes the alleviation or prevention of symptoms, slowing or stoppingthe progression or worsening of a disease, disorder, or condition and/orthe remission of the disease, disorder or condition. “Treatment” refersto both therapeutic treatment and prophylactic or preventative measures.Those in need of treatment include those already affected by a diseaseor disorder or undesired physiological condition as well as those inwhich the disease or disorder or undesired physiological condition is tobe prevented.

In the methods of the present invention, the term “control” andgrammatical variants thereof, are used to refer to the prevention,partial or complete inhibition, reduction, delay or slowing down of anunwanted event, such as the presence or onset of Huntington's disease.

The term “effective amount” refers to an amount sufficient to effectbeneficial or desirable clinical results.

The term “flag-tagged” when used herein refers to a chimeric polypeptidecomprising a single-chain variable region fragment Ab (scFv) fused to a“flag epitope.” The flag epitope has enough residues to provide anepitope against which an antibody may bind for detection purposes(Chiang et al., Pept. Res., 6:62-64 (1993)), but is also short enoughsuch that it does not interfere with the activity of the scFv to whichit is fused.

Antibodies to Huntingtin

Preferred antibodies are specific for particular epitopes on thehuntingtin protein. The huntingtin protein comprises apolyglutamine-rich region close to the N-terminus of the protein, anadjacent polyproline-rich region and a carboxy-terminus region that ischaracterized by the sequence of SEQ ID NO: 2. DNA encoding theglutamine- and proline-rich regions of the human huntingtin protein arecharacterized by a polymorphic trinucleotide repeats. In particular, thepolyglutamine region comprises a number of CAG repeats, encoding forglutamine residues. The CAG repeats are expanded on disease chromosomes.The adjacent polyproline region comprises polymorphic trinucleotide CCGrepeats, encoding for prolines.

In the human huntingtin gene, the polymorphic CAG repeat region variesfrom 13 to 36 repeats and is encoded almost entirely by CAG. The mousehuntingtin gene encodes 7 consecutive glutamine residues in an imperfectrepeat. In both species, the glutamine-rich region is followed by asegment with runs of prolines with interspersion of an occasionalglutamine or other amino acid residue (Rubinsztein et al., Nat. Genet.,5(3):214-5 (1993), incorporated herein by reference). The polyprolineregions of the huntingtin protein are well defined and found, forexample, in SEQ ID NO: 5 in U.S. Pat. No. 5,693,757. These polyprolineregions have sequences of at least 10 consecutive proline residues inthe wild-type sequence.

More specifically, the preferred antibodies recognize an epitope withinthe polyglutamine-rich, polyproline-rich or carboxy-terminus domains ofthe huntingtin protein. By “recognize” it is meant that the antibodiesbind to the huntingtin protein at the particular epitope. In manyembodiments, the subject antibodies do not bind to any appreciableextent to proteins that do not share a significant degree of homologywith the huntingtin protein.

The epitope specificity of the antibodies can be determined by epitopemapping as described, for example, in Ko et al., Brain ResearchBulletin, 56:319-329 (2001) and in the Examples below.

Antibodies are preferably prepared by standard methods well-known in theart. The subject antibody compositions may be polyclonal, such that aheterogeneous population of antibodies differing by specificity ispresent, or monoclonal, in which a homogeneous population of identicalantibodies that have the same specificity for the polyproline region ofthe huntingtin protein are present. As such, both monoclonal andpolyclonal antibodies are provided by the subject invention. In manypreferred embodiments, the subject antibodies are monoclonal antibodies.Specific monoclonal antibodies of interest include: MW1, MW2, MW7, MW8and hMW9, where MW stands for “Milton Wexler,” and are encoded by thenucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQID NO: 6, respectively.

Generally, an antigen or immunogen that can elicit an immune responsecharacterized by the presence of antibodies of the subject invention isemployed. The immunogen preferably comprises at least includes a portionof a protein having a polyglutamine repeat region.

In one embodiment, the immunogen is at least a portion of a wild-type ormutant huntingtin protein, comprising a polyglutamine region having atleast 19 glutamine repeats. The portion of the wild-type or mutanthuntingtin protein may comprise exon 1 of the huntingtin protein,referred to herein as “HDx-1.” A preferred HD-1x immunogen has thesequence of SEQ ID NO: 1, and comprises a polyglutamine region, apolyproline region and a carboxy-terminus region characterized by aneight amino acid stretch having the sequence AEEPLHRP (SEQ ID NO: 2).

In another embodiment, the immunogen is at least a portion of thewild-type or mutant dentatorubral palliodoluysian atrophy (DRPLA)protein (Onodera et al., FEBS Lett., 399:135-139 (1996)). The DRPLAprotein preferably comprises a polyQ domain having from 19 to 35glutamine repeats.

In the preferred embodiments, the immunogen is present in its aggregatedstate. In certain embodiments, other domains are also present in theimmunogens. For example, a glutathione-S-transferase domain may bepresent in the immunogen (Onodera et al., FEBS Lett., 399:135-139(1996); Harris, Methods Mol Biol, 88:87-99 (1998)). Other domains may beincluded. For example, domains may be included that serve to facilitatepurification and identification of the antigen of interest. Theimmunogen is typically employed in the preparation of the subjectantibodies as follows.

Although methods of making monoclonal and polyclonal antibodies are wellknown in the art, preferred methods are briefly described herein.Variations of the following methods will be apparent to one of skill inthe art.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with the immunogen. To increase the immune responseof the host animal, the immunogen may be combined with an adjuvant.Suitable adjuvants include alum, dextran, sulfate, large polymericanions, oil & water emulsions, e.g. Freund's adjuvant, Freund's completeadjuvant, and the like. The immunogen may also be conjugated tosynthetic carrier proteins or synthetic antigens. A variety of hosts maybe immunized to produce the polyclonal antibodies. Such hosts includewithout limitation, rabbits, guinea pigs, other rodents such as mice orrats, sheep, goats, primates and the like. The immunogen is administeredto the host, usually intradermally, with an initial dosage followed byone or more, usually at least two, additional booster dosages. Followingimmunization, the blood from the host is collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

As with the preparation of polyclonal antibodies, the first step inpreparing monoclonal antibodies specific for an epitope within thehuntingtin protein, is to immunize a suitable host. Suitable hostsinclude rats, hamsters, mice, monkeys and the like, and are preferablymice. Monoclonal antibodies may be generated using the hybridoma methoddescribed by Kohler et al., Nature, 256:495 (1975) or by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567.

The immunogen is administered to the host in any convenient manner knownin the art. For example, and without limitation, administration may beby subcutaneous injection with adjuvants, nitrocellulose implantscomprising the immunogen or intrasplenic injections. Alternatively,lymphocytes may be immunized in vitro. The immunization protocol may bemodulated to obtain a desired type of antibody, e.g. IgG or IgM, wheresuch methods are known in the art (Kohler and Milstein, Nature, 256:495(1975)). Booster immunizations may be made, for example one month afterthe initial immunization. Animals are bled and analyzed for antibodytiter. Boosting may be continued until antibody production plateaus.

Following immunization, plasma cells are harvested from the immunizedhost. Sources of plasma cells include the spleen and lymph nodes, withthe spleen being preferred.

The plasma cells are then immortalized by fusion with myeloma cells toproduce hybridoma cells. Fusion may be carried out by an electrocellfusion process or by using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-109, [Academic Press, 1996]). The plasmaand myeloma cells are typically fused by combining the cells in a fusionmedium usually in a ratio of about 10 plasma cells to 1 myeloma cell,where suitable fusion mediums include a fusion agent, e.g. PEG 1000, andthe like. Following fusion, the fused cells will be selected, e.g. bygrowing on HAT medium.

A variety of myeloma cell lines are available. Preferably, the myelomacell is HGPRT negative, incapable of producing or secreting its ownantibodies, and growth stable. Preferred myeloma cells also fuseefficiently and support stable high-level production of antibody by theselected antibody-producing cells. Among these, preferred myeloma celllines are murine myeloma lines, such as those derived from MOP-21 andMC.-11 mouse tumors available from the Salk Institute Cell DistributionCenter, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells availablefrom the American Type Culture Collection, Rockville, Md. USA. Humanmyeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63, Marcel Dekker, Inc.,New York, [1987]). Specific cell lines of interest include, for example,p3U1, SP 2/0 Ag14, P3.times.63Ag8.653 (Dr. Greenberg, V.A. Hospital).

Representative hybridomas according to the subject invention includethose hybridomas that secrete one of the following monoclonalantibodies: MW1, MW2, MW7, MW8 and hMW9. Each of these antibodies isdescribed in detail below.

Following hybridoma cell production, culture supernatant from individualhybridomas is screened for reactivity with huntingtin protein,particularly mutant huntingtin protein, using standard techniques. Suchscreening techniques are well known in the art and includeradioimmunoassay (RIA), enzyme-linked immunosorent assay (ELISA), dotblot immunoassays, Western blots and the like. The binding affinity ofthe monoclonal antibody may, for example, be determined by the Scatchardanalysis (Munson et al., Anal. Biochem., 107:220 (1980)).

After hybridoma cells secreting antibodies with the desired specificity,affinity and/or activity are selected, the cells may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103, AcademicPress, 1996). Culture media may be for example DMEM or RPMI-1640 medium.Alternatively, hybridomas may be grown in vitro as ascites tumors in ananimal.

The desired antibody may be purified from the supernatants or ascitesfluid by conventional techniques, e.g. affinity chromatography usingmutant huntingtin protein bound to an insoluble support, protein Asepharose and the like.

DNA encoding the monoclonal antibody may be isolated and sequenced usingconventional procedures, with the hybridoma cells serving as a source ofthe DNA. The isolated DNA may be introduced into host cells in cultureto synthesize the monoclonal antibodies in the recombinant host cells.The DNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences, Morrison, et al., Proc. Nat. Acad. Sci.81, 6851 (1984), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In that manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of an anti-Huntingtin proteindescribed herein.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,J. Immunol. 133, 3001 (1984), and Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90, 2551-255 (1993); Jakobovits et al.,Nature 362, 255-258 (1993).

Mendez et al. (Nature Genetics 15: 146-156 [1997]) have further improvedthe technology and have generated a line of transgenic mice designatedas “Xenomouse II” that, when challenged with an antigen, generates highaffinity fully human antibodies. This was achieved by germ-lineintegration of megabase human heavy chain and light chain loci into micewith deletion into endogenous J_(H) segment as described above. TheXenomouse II harbors 1,020 kb of human heavy chain locus containingapproximately 66 V_(H) genes, complete D_(H) and J_(H) regions and threedifferent constant regions (μ, δ and χ), and also harbors 800 kb ofhuman κ locus containing 32 Vκ genes, Jκ segments and Cκ genes. Theantibodies produced in these mice closely resemble that seen in humansin all respects, including gene rearrangement, assembly, and repertoire.The human antibodies are preferentially expressed over endogenousantibodies due to deletion in endogenous J_(H) segment that preventsgene rearrangement in the murine locus.

Alternatively, phage display technology (McCafferty et al., Nature 348,552-553 [1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors.

Binding fragments or binding mimetics of the subject antibodies may alsobe prepared. These fragments and mimetics preferably share the bindingcharacteristics of the subject antibodies. “Binding characteristics”when used herein include specificity, affinity, avidity, etc. for thehuntingtin protein, particularly the polyglutamine, polyproline orc-terminal region of exon 1. The subject antibodies are modified tooptimize their utility, for example for use in a particular immunoassayor their therapeutic use. In one embodiment antibody fragments, such asFv and Fab may be prepared by cleavage of the intact protein, e.g. byprotease or chemical cleavage. Nucleic acid encoding the antibodyfragments or binding mimetics may be identified.

Antibody fragments, such as single chain antibodies or scFvs, may alsobe produced by recombinant DNA technology where such recombinantantibody fragments retain the binding characteristics of the aboveantibodies. “Antibody fragments” when used herein refer to a portion ofan intact antibody, such as the antigen binding or variable region andmay include single-chain antibodies, Fab, Fab′, F(ab′)2 and Fvfragments, diabodies, linear antibodies, and multispecific antibodiesgenerated from portions of intact antibodies.

Recombinantly produced antibody fragments generally include at least theV_(H) and V_(L) domains of the subject antibodies, so as to retain thedesired binding characteristics. These recombinantly produced antibodyfragments or mimetics may be readily prepared from the antibodies of thepresent invention using any convenient methodology, such as themethodology disclosed in U.S. Pat. Nos. 5,851,829 and 5,965,371; thedisclosures of which are herein incorporated by reference. The antibodyfragments or mimetics may also be readily isolated from a human scFvsphage library (Pini et al., Curr. Protein Pept. Sci., 1(2):155-69(2000)) using huntingtin protein, particularly mutant huntingtinprotein.

The invention also provides isolated nucleic acid encoding theanti-huntingtin antibodies, vectors and host cells comprising thenucleic acid, and recombinant techniques for the production of theantibodies.

For recombinant production of an antibody, the nucleic acid encoding itmay be isolated and inserted into a replicable vector for furthercloning and expression. DNA encoding the antibody is readily isolatedand sequenced using conventional procedures. Many cloning and expressionvectors are available and are well known in the art. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence, e.g., as described in U.S. Pat. No. 5,534,615.

Host cells, preferably eukaryotic cells such as CHO cell or COS cells,are transformed with the above-described expression or cloning vectorsfor anti-huntingtin antibody production and cultured according towell-established procedures.

Screening for Antibodies with Desired Properties

Once antibodies to the immunogen have been produced, they may bescreened for desirable biological properties, such as high affinitybinding to the desired antigen, specific binding to particular mutantforms of the huntingtin protein, the ability to prevent cell death orapoptosis associated with mutant huntingtin protein, and/or the abilityto prevent aggregation of mutant huntingtin protein.

Prevention of Cell Death or Apoptosis

In a preferred embodiment, antibodies are identified that reduce thelevel of cell death associated with expression of mutant huntingtinprotein. This activity may be observed in a model system forHuntington's disease, for example using terminaldeoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)staining. Such an assay is described in Example 2, below and in Khoshnanet al., PNAS, 99:1002-1007 (2002), the entire contents of which areincorporated herein by reference in its entirety.

Specifically, extensive cellular DNA degradation is a characteristicevent which often occurs in the early stages of apoptosis and ismediated by a Ca2+-dependent endonuclease. As cleavage of the DNA inapoptotic cells results in double-stranded DNA fragments and singlestrand breaks, the degraded DNA may be detected by labeling methods. Forexample, enzymatic labeling of the free 3′-OH termini of the cellularDNA with modified nucleotides using exogenous enzymes, such as terminaldeoxynucleotidyl transferase, is used to detect DNA strand breaks. Thelabeled DNA may be subsequently analyzed by immunocytochemistry (ICC),such as flow cytometry, fluorescence microscopy or light microscopy.Accordingly, preferred antibodies that reduce the level of apoptosis ina model system for Huntington's disease may be selected using the TUNELstaining.

Prevention of Mutant Huntingtin Aggregation

In a preferred embodiment of the invention, anti-huntingtin antibodies,particularly those that are directed to the polyproline region, areidentified that have the ability to inhibit the aggregation ofhuntingtin in vivo. Aggregation of the huntingtin protein is associatedwith Huntington's disease and is present in affected neurons.Aggregation may be evaluated by examining the amount of huntingtinprotein that is precipitated from cell lysates by centrifugation. Theamount of aggregation is analyzed by subjecting the lysates that weresubjected to centrifugation to SDS-PAGE. An exemplary assay is describedin Example 2 and in Khoshnan et al., PNAS, 99:1002-1007 (2002).

Diagnostic Applications

The subject antibodies, binding fragments and mimetics thereof find usein immunoassays that are capable of providing for the detection ofhuntingtin or mutant huntingtin protein in a sample. In such assays, thesample suspected of comprising the huntingtin or mutant huntingtinprotein of interest will typically be obtained from a subject, such as ahuman subject, suspected of suffering from the disease of interest or atrisk for developing the disease of interest. The sample is generally aphysiological sample from the patient such as blood or tissue. Dependingon the nature of the sample, it may or may not be pretreated prior toassay, as will be apparent to one of skill in the art.

A number of different immunoassay formats are known in the art and maybe employed in detecting the presence of protein of interest in asample. Immunoassays of interest include Western blots on protein gelsor protein spots on filters, where the antibody is labeled, as is knownin the art. A variety of protein labeling schemes are known in the artand may be employed, the particular scheme and label chosen being theone most convenient for the intended use of the antibody, e.g.immunoassay. Examples of labels include labels that permit both thedirect and indirect measurement of the presence of the antibody.Examples of labels that permit direct measurement of the antibodyinclude radiolabels, such as ³H or ¹²⁵I, fluorescent dyes, beads,chemiluminescers and colloidal particles. Examples of labels whichpermit indirect measurement of the presence of the antibody includeenzymes where a substrate may provided for a colored or fluorescentproduct. For example, the antibodies may be labeled with a covalentlybound enzyme capable of providing a detectable product signal afteraddition of suitable substrate. Instead of covalently binding the enzymeto the antibody, the antibody may be modified to comprise a first memberof specific binding pair which specifically binds with a second memberof the specific binding pair that in conjugated to the enzyme, e.g. theantibody may be covalently bound to biotin and the enzyme conjugate tostreptavidin. Examples of suitable enzymes for use in conjugates includehorseradish peroxidase, alkaline phosphatase, malate dehydrogenase andthe like. Where not commercially available, such antibody-enzymeconjugates are readily produced by techniques known to those skilled inthe art.

Other immunoassays include those based on a competitive formats, as areknown in the art. One such format would be where a solid support iscoated with the polyproline region containing protein, including forexample the mutant huntingtin protein. Labeled antibody is then combinedwith a sample suspected of comprising protein of interest to produce areaction mixture which, following sufficient incubation time for bindingcomplexes to form, is contacted with the solid phase bound protein. Theamount of labeled antibody which binds to the solid phase will beproportional to the amount of protein in the sample, and the presence ofprotein may therefore be detected. Other competitive formats that may beemployed include those where the sample suspected of comprising proteinis combined with a known amount of labeled protein and then contactedwith a solid support coated with antibody specific for the protein. Suchassay formats are known in the art and further described in both Guideto Protein Purification, supra, and Antibodies, A Laboratory Manual(Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989)).

In immunoassays involving solid supports, the solid support may be anycompositions to which antibodies or fragments thereof can be bound,which is readily separated from soluble material, and which is otherwisecompatible with the overall immunoassay method. The surface of suchsupports may be solid or porous and of any convenient shape. Examples ofsuitable insoluble supports to which the receptor is bound includebeads, e.g. magnetic beads, membranes and microtiter plates. These aretypically made of glass, plastic (e.g. polystyrene), polysaccharides,nylon or nitrocellulose. Microtiter plates are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

Before adding patient samples or fractions thereof, the non-specificbinding sites on the insoluble support i.e. those not occupied by thefirst antibody, are generally blocked. Preferred blocking agents includenon-interfering proteins such as bovine serum albumin, casein, gelatin,and the like. Alternatively, detergents, such as Tween, NP40 or TX100may be used at non-interfering concentrations.

It is particularly convenient in a clinical setting to perform theimmunoassay in a self-contained apparatus, and such devices are providedby the subject invention. A number of such devices and methods for theiruse are known in the art. The apparatus will generally employ acontinuous flow-path over a suitable filter or membrane, and will haveat least three regions, a fluid transport region, a sample region, and ameasuring region. The sample region is prevented from fluid transfercontact with the other portions of the flow path prior to receiving thesample. After the sample region receives the sample, it is brought intofluid transfer relationship with the other regions, and the fluidtransfer region contacted with fluid to permit a reagent solution topass through the sample region and into the measuring region. Themeasuring region may have bound to it a first antibody. The second,labeled antibody combined with the assayed sample is introduced and thesandwich assay performed as above.

Screening to Identify Compounds with a Desired Biological Activity

The subject antibodies, binding fragments and mimetics thereof also finduse in screening applications designed to identify agents or compoundsthat are capable of modulating, e.g. inhibiting, the binding interactionbetween the protein to which the antibody binds and a cellular target.For example, the subject antibodies find use in screening assays thatidentify compounds capable of modulating the interaction between mutanthuntingtin protein and its cellular targets. In such assays, the subjectantibody is contacted with mutant huntingtin protein in the presence ofa candidate modulation agent and any resultant binding complexes betweenthe antibody and the mutant huntingtin protein are detected. The resultsof the assay are then compared with a control. Those agents which changethe amount of binding complexes that are produced upon contact areidentified as agents that modulate the binding activity of mutanthuntingtin protein and therefore are potential therapeutic agents. Ofinterest in many embodiments is the identification of agents thatinhibit, at least to some extent, the binding of mutant huntingtinprotein with its target. In many assays, at least one of the protein orantibody is attached to a solid support and at least one of thesemembers is labeled, where supports and labels are described supra.

In other assays, the ability of a candidate compound to disrupt orenhance the biological activity of an anti-huntingtin antibody ismeasured. For example, the ability of a candidate compound to prevent orenhance the inhibition of cell death, apoptosis or aggregation normallyproduced by an anti-huntingtin antibody may be measured.

A variety of different candidate agents may be screened by the abovescreening methods. Candidate agents encompass numerous chemical classes,though typically they are organic molecules, preferably small organiccompounds having a molecular weight of more than 50 and less than about2,500 daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Methods of Treatment

An individual suffering from Huntington's disease may be treated usingantibodies of the present invention or compounds identified in screensusing the antibodies. By treatment is meant at least an amelioration ofthe symptoms associated with the pathological condition afflicting thehost, where amelioration is used in a broad sense to refer to at least areduction in the magnitude of a parameter, e.g. symptom, associated withthe pathological condition being treated, such as neuronal cell death.As such, treatment includes situations where the pathological condition,or at least symptoms associated therewith, are completely inhibited,e.g. prevented from happening, or stopped, e.g. terminated, such thatthe host no longer suffers from the pathological condition, or at leastthe symptoms that characterize the pathological condition.

A variety of individuals are treatable according to the subject methods.Generally such individuals are “mammals” or “mammalian,” where theseterms are used broadly to describe organisms which are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the individuals will behumans.

In certain embodiments, the methods of treatment involve administrationof an effective amount of a compound that modulates, e.g. inhibits, theinteraction of a mutant huntingtin protein, with its cellular targets.The compound is preferably an antibody of the invention that targets thepolyproline region of the huntingtin protein, the polyglutamine regionof the huntingtin protein or an epitope within the c-terminal sequenceof exon 1 of the huntingtin protein. In a preferred embodiment theantibodies are human or humanized, such that any undesirable immuneresponse in the patient is minimized.

The anti-huntingtin antibodies may be administered using any convenientprotocol capable of resulting in the desired therapeutic activity. Thus,the agent can be incorporated into a variety of formulations fortherapeutic administration. More particularly, the agents of the presentinvention can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents (Remington: The Science and Practice of Pharmacy, 19^(th)Edition, Alfonso, R., ed., Mack Publishing Co. (Easton, Pa.: 1995)), andmay be formulated into preparations in solid, semi-solid, liquid orgaseous forms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols.

Anti-huntingtin protein antibodies can also be administered byinhalation. Commercially available nebulizers for liquid formulations,including jet nebulizers and ultrasonic nebulizers are useful for suchadministration. Anti-huntingtin protein antibodies can also beaerosolized using a fluorocarbon formulation and a metered dose inhaler,or inhaled as a lyophilized and milled powder.

The anti-huntingtin antibodies to be used for in vivo administrationmust be sterile. The sterility may be accomplished by filtration usingsterile filtration membranes, prior to or following lyophilization andreconstitution. The anti-huntingtin antibodies may be stored inlyophilized form or in solution.

The anti-huntingtin antibody compositions may be placed into a containerwith a sterile access port, for example, an intravenous solution bag orvial having a stopper pierceable by a hypodermic injection needle.

In pharmaceutical dosage forms, the antibodies or other compounds may beused alone or in appropriate association, as well as in combination withother pharmaceutically active or inactive compounds. The followingmethods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Each dosage for human and animal subjects will preferably contain apredetermined quantity of compounds of the present invention calculatedin an amount sufficient to produce the desired effect, in associationwith a pharmaceutically acceptable diluent, carrier or vehicle. Thespecifications for the novel unit dosage forms of the present inventiondepend on the particular compound employed and the effect to beachieved, and the pharmacodynamics associated with each compound in thehost.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, antioxidants, low molecular weight (less thanabout 10 residues) polypeptides, tonicity adjusting agents, stabilizers,wetting agents and the like, are readily available to the public.“Carriers” when used herein refers to pharmaceutically acceptablecarriers, excipients or stabilizers which are nontoxic to the cell ormammal being exposed to the carrier at the dosages and concentrationsused.

Administration of the agents can be achieved in various ways, includingintracranial, either injected directly into the brain tissue or injectedinto the cerebrospinal fluid, oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intracheal, intracerebral,etc., administration. The antibodies may be administered in combinationwith one or more additional therapeutic agents. Administration may bechronic or intermittent, as deemed appropriate by the supervisingpractitioner, particularly in view of any change in the disease state orany undesirable side effects. Administration “in combination with” oneor more further therapeutic agents includes both simultaneous (at thesame time) and consecutive administration in any order. “Chronic”administration refers to administration of the agent in a continuousmanner while “intermittent” administration refers to treatment that isnot done without interruption.

In a particular embodiment, antibodies of the invention are administeredby intracranial injection. The injection will typically be directly intoaffected brain regions or into the cerebrospinal fluid.

An effective amount of an antibody or compound of the present inventionto be employed therapeutically will depend, for example, upon thetherapeutic objectives, the route of administration, and the conditionof the patient. Accordingly, it will be necessary for the therapist totiter the dosage and modify the route of administration as required toobtain the optimal therapeutic effect. A typical daily dosage mightrange from about 1 μg/kg to up to 100 mg/kg or more, depending on thefactors mentioned above. Typically, the clinician will administer amolecule of the present invention until a dosage is reached thatprovides the required biological effect. The progress of this therapy iseasily monitored by conventional assays.

Also provided by the subject invention are methods of treatingHuntington's disease conditions by expressing antibodies, particularlyintrabodies, i.e. non-secreted forms of the subject antibodies, e.g.scFv analogs of the subject antibodies, as described supra, in cellsexpressing mutant huntingtin protein. Intrabodies and methods for theiruse in the treatment of disease conditions are described in U.S. Pat.Nos. 5,851,829 and 5,965,371, the disclosures of which are hereinincorporated by reference. In such methods, a nucleic acid encoding theantibody or intrabody, generally in the form of an expression cassettethat includes a sequence encoding the antibody domains of interest, suchas the V_(H) and V_(L) domains, as well as other components, e.g.promoters, linkers, intracellular localization domains or sequences,etc., is introduced into the target cells in which antibody or intrabodyproduction is desired. The nucleic acid is introduced into the targetcells using any convenient methodology, e.g. through use of a vector,such as a viral vector, liposome vector, by biolistic transfection andthe like, where suitable vectors are well known in the art. Viral and/ornon-viral methods of delivering the nucleic acid encoding the intrabodyto the cell may be used.

A wide variety of non-viral vehicles for delivery of a polynucleotideencoding an antibody of the present invention are known in the art andare encompassed in the present invention. A nucleic acid encoding ananti-huntingtin antibody or intrabody can be delivered to a cell asnaked DNA (U.S. Pat. No. 5,692,622; WO 97/40163). Alternatively, a thenucleic acid can be delivered to a cell by association with one or moreof a variety of substances including, but not limited to cationiclipids; biocompatible polymers, including natural polymers and syntheticpolymers; lipoproteins; polypeptides; polysaccharides;lipopolysaccharides; artificial viral envelopes; metal particles; andbacteria. The nucleic acid could also be delivered as a microparticle.Mixtures or conjugates of these various substances can also be used asdelivery vehicles. The nucleic acid can be associated non-covalently orcovalently with these delivery agents. It is possible to targetliposomes to a particular cell type.

Viral vectors include, but are not limited to, DNA viral vectors such asthose based on adenoviruses, herpes simplex virus, poxviruses such asvaccinia virus, and parvoviruses, including adeno-associated virus; andRNA viral vectors, including, but not limited to, retroviral vectors.Retroviral vectors include, for example, murine leukemia virus, andlentiviruses such as human immunodeficiency virus. Naldini et al.,Science 272:263-267 (1996).

In a particular embodiment, the nucleic acid encoding the intrabody thatis to be expressed is inserted into a viral vector. Preferred viralconstructs are based on a retroviral genome, more preferably alentiviral genome as these viruses are able to infect both dividing andnon-dividing cells. The vector is transfected into packaging cells andrecombinant retrovirus is collected. The recombinant retrovirus is thencontacted with the cells in which expression of the intrabody isdesired. For example, the virus may be injected intracranially or intothe cerebrospinal fluid. In a particular embodiment, the virus isinjected directly into brain regions that are known to be affected byHuntington's disease.

Following introduction of the nucleic acid into the target cells, thenucleic acid is allowed to be expressed in the target cell, wherebyintrabodies that specifically bind to the protein of interest, e.g.mutant huntingtin protein, are produced in the cell. Production of theintrabodies interferes with the activity of the protein, e.g. mutanthuntingtin protein, and thereby treats the host suffering from thedisease condition.

EXAMPLES

Further details of the invention can be found in the following example,which further defines the scope of the invention. The followingexamples, including the experiments conducted and achieved are providedfor illustrative purposes only and are not to be construed as limitingupon the present invention. All references cited throughout thespecification, are hereby expressly incorporated by reference in theirentirety.

Example 1 Anti-Huntingtin Antibodies

A. Production of Anti-Huntingtin Antibodies

The antigens used and the isotypes of the MW monoclonal anti-huntingtinantibodies are summarized in Table 1.

1. Immunization

For generation of anti-huntingtin antibodies, six-week-old Balb/c femalemice were primed and boosted at 2 week intervals by intraperitonealinjection of antigen emulsified in adjuvant (RIBI Immunochem, Hamilton,Mont., USA). Three different methods were used for the generation of theanti-huntingtin antibodies.

a. DRPLA-19Q or DRPLA-35Q

For generation of mAbs, herein referred to as MW (for Milton Wexler)mAbs, MW1, MW2 and MW5, mice were injected with antigen proteins thatwere expressed from two constructs comprising the polyQ domain (19 or 35repeats) of huntingtin and 34 amino acids of thedentatorubralpalliodoluysian atrophy (DRPLA) gene fused toglutathione-S-transferase (GST) (Onodera et al., FEBS Lett, 399:135-139(1996)). Test bleeds were obtained 7 days after very other injection. Afinal series of boosts were performed without adjuvant. Spleen cellswere isolated from the mouse 3 days after the final boost and fused withHL-1 murine myeloma cells (Ventrex, Portland, Me. USA) usingpolyethylene glycol (PEG 1500, Boehringer-Mannheim, Mannheim, Germany)(Lebron et al., J. Immunol., 222:59-63 (1999)). Using enzyme linkedsubstrate assay (ELISA) to screen against these antigens versus GSTalone, three hybridomas were selected for cloning.

b. Expanded PolyQ Domain of Exon 1 of Huntingtin Protein in Soluble Form

For generation of mAbs, MW3, MW4 and MW6, mice were immunized asdescribed above with protein that was soluble in aqueous solution andwas expressed from a construct comprising the expanded polyQ domain (67glutamine repeats) of Htt exon 1 (67Q) fused to GST (GST-HDx67Q). Spleencells were isolated from the mice 3 days after the final boost and fusedwith HL-1 murine myeloma cells.

c. Expanded PolyQ Domain of Exon 1 of Huntingtin Protein in Soluble andAggregated Form

For generation of mAbs, MW7 and MW8, mice were immunized with the sameHtt exon 1 (67Q) protein fused to GST (GST-HDx67Q). However, boosting ofthe mice was performed with an aggregated form of exon 1 having 67 Qrepeats of the huntingtin protein (67Q), prepared by removing the GST.Spleen cells were isolated from the mice 3 days after the final boostand fused with HL-1 murine myeloma cells.

2. Selection of Hybridomas

Three hybridomas generated from mice immunized with proteins expressedfrom the two constructs containing the polyQ domain (19 or 35 repeats)and 34 amino acids of the dentatorubralpalliodoluysian atrophy (DRPLA)gene fused to glutathione-S-transferase (GST) were selected for cloning.mAbs from these hybridomas were termed MW1, MW2, and MW5.

The hybridomas generated from mice immunized with GST-HDx67Q, and frommice immunized with the same GST-HDx67Q antigen and boosted with anaggregated form that lacked the GST were both screened by ELISA usingthe antigen, GST-HDx67Q and GST alone, and by Western blotting ofextracts from the Huntington's disease (HD) lymphoblastoma cell lineHD2. mAbs, MW3, MW4 and MW6 were generated from hybridomas generatedfrom mice immunized with GST-HDx67Q while mAbs, MW7 and MW8 weregenerated from hybridomas generated from mice immunized with GST-HDx67Qand boosted with an aggregated form of GST-HDx67Q that lacked GST.

a. ELISA

The hybridomas were analyzed by ELISA using the antigen, GST-HDx67Q andGST alone. MW3, MW4 and MW6 bound the injected protein, GST-HDx67Q, butdid not bind to GST alone. MW7 and MW 8 were selected for having apositive ELISA signal with GST-HDx67Q.

b. Western Blots

For the Western blots, lymphoblasts from control (HD7) and HD patients(HD2) were cultured in Isscove's modified Dulbecco's medium (IrvineScientific, Irvine, Calif. USA) supplemented with 15% fetal calf serumand 2 mM glutamine. Lymphoblasts or cerebella from mice were homogenizedin 300 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 50 mM Tris, pH 7.0, withcomplete protease inhibitor cocktail (Boehringer Mannheim). Thehomogenates were centrifuged (14,300 rpm for 10 min). The proteinconcentrations of the supernatants were determined by BCA assay (Pierce,Rockford, Ill., USA). The protein in the supernatants were concentratedby precipitation at 70° C. for 3 min. The precipitates were resuspendedin 6 M urea at one half of the original supernatant volume andconcentrated sodium dodecyl sulfate (SDS) dissociation buffer added toachieve a final concentration of 5% 2-mercaptoethanol, 1.5% SDS, and 5%glycerol. Samples were heated at 95° C. for 10 minutes and subjected toSDS polyacrylamide gel electrophoresis (PAGE) on 5% gels (Laemmlie, E.K., Nature, 227:105-132 (1970)). Gels were electrotransferred tonitrocellulose membrane (Schleicher & Schuell, Keene, N.H., USA)overnight with cooling. These membranes were then preblocked with 1%blocking reagent (Boehringer Mannheim) and incubated with the MW mAbs(undiluted hybridoma supernatants), MAB2166 (Chemicon, Temecula, Calif.,USA; 1/1000 dilution), 1F8 ascites fluid (M. MacDonald; 1/1000dilution), overnight at room temperature. Blots were washed with 0.5%Tween-20 in phosphate-buffered saline (PBS) for 10 minutes 3 timesbefore incubation with biotinylated goat anti-mouse immunoglobulin (Ig)G+IgM (Chemicon), diluted 1/1000 in blocking buffer, for 1 hour at roomtemperature. After washing, the blots were incubated with horseradishperoxidase-strepavidin (Chemicon) in blocking buffer for 1 hour at roomtemperature. Blots were developed using 4-chloro-1-naphthol. TABLE 1GENERATION AND CHARACTERIZATION OF ANTI-HUNTINGTIN (Htt) MONOCLONALANTIBODIES (mAbs) Antigen MAb Isotype Epitope Immunoblot ICC DRPLA-19QMW1 IgG2b polyQ Mutant Htt Cytoplasm DRPLA-35Q and MW2 IgM polyQ MutantHtt Golgi TRX-35Q MW5 IgM polyQ Mutant Htt + other Golgi bands HDx-67Q(soluble) MW3 IgM polyQ Mutant Htt + other Golgi bands MW4 IgM polyQMutant Htt + other Golgi bands MW6 IgM polyQ Band below 350 kD +Cytoplasm variable size band HDx-67Q (soluble 1^(st) MW7 IgM polyP 350kD + 130 kD Perinuclear in wild-type and boost with and lower mousebrain; inclusions aggregate) in R6/2 brain MW8 IgG2a AEEPLHRPK ? Nostaining in wild-type mouse brain; inclusions in R6/2 brain

B. Characterization of Anti-Htt Antibodies

1. Epitope Mapping

To determine the epitopes recognized by these mAbs, we utilized arraysof dot blots that contain overlapping 14mer peptides synthesized fromthe first 91 amino acids of normal human Htt (containing a 23 polyQdomain). The first dot contained the peptide corresponding to aminoacids 1-14, the second dot contained the peptide corresponding to 4-17,the third dot contained the peptide corresponding to 7-20, etc.

Each of the MW1-MW6 mAbs specifically bound one of three single,contiguous epitopes in the Htt sequence (FIG. 1). MW1-MW6 bound peptidesthat contain >6 glutamines and were specific for the polyQ region. Asthe antigens used to generate the MW1-MW6 mAbs contained other aminoacids in numbers equal or greater than the polyQ domain, the polyQdomain may be highly antigenic or may be prominently displayed insoluble protein fragments.

As summarized in FIG. 2, MW7 specifically binds peptides that containthe polyP domain in Htt. There are two of these domains in exon 1, andMW7 binds all peptides with >5 consecutive prolines. MW8, in contrast,binds specifically to an eight amino acid stretch, AEEPLHRP (SEQ ID NO:2), near the Cterminus of exon 1. MW7 and MW8 mAbs did not bind thepolyQ domain in Htt.

2. Western Blots

To determine if MW1-MW8 mAbs were able to distinguish between normal andmutant Htt containing the expanded polyQ, the MW1-MW8 mAbs were testedin parallel for binding, as analyzed by immunoblotting, to brainextracts of a wild-type mouse and a knock-in transgenic mouse thatexpresses a mouse human chimeric Htt exon 1 construct that contains 94Qrepeats (94Q knock-in mouse) (Menalled et al., Exp. Neurol., 162:328-342(2000)) (FIG. 3) and extracts of a lymphoblastoma cell line from a humanHD patient (HD2) that expressed both normal and mutant Htt and alymphoblastoma cell line from human non-HD patient (HD7) that expressedonly normal Htt (FIG. 4). Control antibodies, 1C2 (Chemicon MAb1574;Trottier et al., Nat. Genet., 10:1040110 (1995)) and 1F8 (Wheeler etal., Hum. Mol. Genet., 9:503-513 (2000)) were used to identify mutantHtt, and Ab2166 (Chemicon) were used to identify both mutant and normalHtt.

The extracts used and the procedure for immunoblotting was performed asdescribed above.

MW1-MW6 specifically bound the polyQ epitope in Htt with MW1-MW5preferentially binding to the expanded repeat mutant form of Htt ratherthan normal Htt on Western blots. More specifically, the mAbs MW1 andMW2 displayed a very specific binding pattern similar to the pattern for1C2, strongly staining the expanded mutant polyQ form of Htt that isapproximately 350 kD in size in mouse brain extracts from the 94Q miceand did not bind the normal polyQ form in mouse brain extracts from WTmice (FIG. 3). MW1 and MW2 mAbs also specifically bound the 350 kDa formof mutant Htt in extracts from HD2 cells (FIG. 4).

MW3-MW5 displayed a very specific binding pattern similar to the patternfor 1F8 in mouse brain extracts. MW3-MW5 specifically bound the expandedmutant repeat form of Htt rather than normal Htt as well as other bandsof lower molecular weights which may be breakdown products of Htt withdifferent conformations (FIG. 3). MW3-MW5 mAbs also specifically boundthe 350 kDa form of mutant Htt in extracts from HD2 cells (FIG. 4).

MW6 specifically bound in normal and HD human lymphoblastoma cellextracts an antigen that has a size varying from about 250-300 kDa whichmay be a breakdown product of Htt.

MW7 bound the expanded repeat mutant form of Htt in mouse brain extracts(FIG. 3). In human lymphoblastoma cell extracts, MW7 bound the highmolecular weight Htt very weakly, but binds strongly one or two smallermolecular weight proteins which are present in normal (HD7) andHuntington's disease (HD2) human lymphoblastoma cell extracts (FIG. 4)at roughly equivalent levels.

MW8 did not detectably bind any proteins in mouse brain extracts nor inhuman lymphoblastoma cell extracts examined by immunoblotting.

3. Immunostaining

Light microscopic immunohistochemistry was done with 10-μm sections of4% paraformaldehyde fixed R6/2 tissue or fresh frozen R6/1 tissue.Briefly, 8-10-week-old R6/1, R6/2 or control littermates (JacksonLaboratory; Mangiarini et al., Cell, 87:493-506 (1996)) wereanesthetized with Phenobarbital, perfused with PBS followed by 4%paraformaldehyde or PBS only. Brains were removed and frozen on dry icewith O.C.T. compound (Sakura Finetek, Torrance, Calif., USA). R6/2contains human Htt exon 1 with 144 polyQ repeats while R6/1 contains 116repeats and displays symptoms at a later age than R6/2. Fixed sectionswere incubated with mAbs MW3-8 or 1F8 ( 1/1000). PBS washed sectionswere incubated with Hi-Fluorescence goat anti-mouse IgG (Antibodies,Inc., Davis, Calif., USA) and DTAF goat anti mouse IgG+IgM (Chemicon) inblocking buffer (2% bovine serum albumin, 5% normal goat serum). Freshfrozen sections were incubated with ascites of MW1 or MW2 at 1/1000, or1C2 (Chemicon MAB1574) at 1/1000, in blocking buffer. Biotinylated goatanti-mouse IgG+IgM and fluorescein isothiocyanatestreptavidin were used.Light microscopic images were captured using a digital camera (SPOT,Diagnostic Instruments, Sterling Heights, Mich., USA) attached to anepi-fluorescent microscope (Leica DMLB, Deerfield, Ill., USA).Thirty-five micrometer 4% paraformaldehyde fixed floating sections wereprocessed using the same secondary Abs as above and subjected toconfocal microscopy (Leica DM IRB/E, Leica confocal software).

MW1 and control 1C2 antibodies displayed primarily punctate cytoplasmicstaining of neurons (FIGS. 5B and 5D) in wild-type and R6/2 transgenicbrain sections. Neuropil staining with MW1 which was also apparent wasspecific because controls omitting the primary antibody were largelynegative under the same staining and photographic conditions (FIG. 5 a).

MW2-MW5 and control 1F8 antibodies displayed little or no staining ofthe neuropil, but stained neuronal Golgi complex in wild-type spinalcord section as shown in FIGS. 6A-6E and in R6/2 transgenic spinal cordsections (MW3 staining is shown in FIG. 6F) with no difference instaining between wild-type and mutant transgenic spinal cord withMW3-MW5 antibodies. However, MW3, MW4 and MW5 staining in R6/2 brainsections (MW3 staining is shown in FIG. 6H) was less than staining inwild-type brain sections (MW3 staining is shown in FIG. 6G).

MW6 displayed very strong punctate staining of neuropil and stronghomogeneous staining of neuronal cytoplasm in wild-type (FIG. 7A) andmutant spinal cord (FIG. 7B), with no obvious difference in stainingbetween wild-type and mutant spinal cord. MW6 antibodies did notstrongly stain neuronal nucleus.

MW7 displayed punctate perinuclear or nuclear membrane staining inwild-type (FIG. 7C) and mutant brain sections (FIG. 7D) with weakerpunctate perinuclear or nuclear membrane staining, but more prominentnuclear inclusion staining in mutant R6/2 brain sections. Theperinuclear staining is shown in a confocal microscope series (FIGS.7E-7H).

MW8 mAbs displayed nuclear inclusion staining in R6/2 brain sections(FIG. 7J), but did not stain nuclear inclusions in wild-type brainsections (FIG. 71). MW8 mAbs also stained small inclusions in theneuropil.

In summary, MW1-MW6 mAbs did not stain nuclear inclusions well in brainsections while MW7 and MW8 mAbs stained nuclear inclusions in brainsections of mice expressing a human chimeric Htt exon 1 construct with94Q repeats.

4. Summary

Both the epitope mapping and histochemical results are summarized inFIG. 8. The availability of four regions of exon 1 of Htt, theN-terminal 17 amino acids, the polyQ domain, the polyP domain and theC-terminal domain, for Ab binding was different in the Golgi,perinuclear and nuclear subcellular compartments. The N-terminal 17amino acids of exon 1 of Htt was available for Ab binding in the Golgi,perinuclear and nuclear subcompartments. In the spinal cord neuronalGolgi complex of both WT and R6/2 mice, the polyQ domain was available,but the adjacent polyP and C-terminal domains were occluded. In theperinuclear region of neurons of R6/2 mice, the polyP domain wasavailable for Ab binding, but the C-terminus was occluded. Within thenucleus of neurons in the R6/2 (but not wild-type) mice, the polyQdomain was occluded, but the adjacent N-terminal, C-terminal and polyPdomains were open for Ab binding.

Example 2 Anti-Htt Antibody Fragments

To examine the effects of the anti-huntingtin (Htt) antibodies on thebiological activities of Htt exon 1, we generated single-chain variableregion fragment Abs (scFvs) for MW1 and MW2 anti-Htt antibodies, whichrecognizes the polyQ Http epitope, MW7 anti-Htt antibody, whichrecognizes the polyP domains of Htt exon 1 and MW8 anti-Htt antibody,which recognizes an 8 amino acid epitope near the C-terminus of thehuntingtin protein. Human anti-huntingtin hMW9 antibody was isolatedfrom a human scFvs phage library using recombinant mutant huntingtinprotein. The scFvs for MW1, MW2, MW7 and MW8, expressed in E. coli weretested for binding to Htt on immunoblots, and the scFv for hMW9 weretested for binding to His-HDx in vitro. Positive clones were selectedfor further characterization in mammalian cells.

A. Generation of scFvs

1. MW1, MW2, MW7 and MW8 scFvs

For generation of MW1, MW2, MW7 and MW8 scFvs, total RNA was extractedfrom hybridoma cell lines secreting each of the anti-Htt MW mAbs, andmRNA was purified by using oligo-dT columns (Qiagen, Valencia, Calif.).Complementary cDNA was produced for each mRNA pool by using randomhexanucleotide primers. The cDNAs served as sources of DNA to amplifyboth variable region heavy (VH) and variable region light (VL) chainsfor each mAb by using primers complementary to the consensus sequencesflanking each domain (Amersham Pharmacia) and PCR technology. Togenerate recombinant single-chain fragment Abs, the amplified VH and VLof each mAb were linked by a 45-mer nucleotide encoding Gly-Ser. ThesescFv genes were cloned into the M13 phagemid, pCANTBE5 (AmershamPharmacia), and used to transform Escherichia coli, strain TG15, whichsupports production of recombinant phage. The amplified recombinantphage population was selected for binding on immunoblots to Httexon-1-glutathione S-transferase (GST)-containing a 67-polyQ repeat.Phage that specifically bound Htt were eluted and used to reinfect TG15E. coli. Individual clones were tested again for Htt binding, and thenucleotide sequence of positive clones was determined bydideoxynucleotide chain-termination method. The nucleotide sequence ofMW1, MW2, MW7 and MW8 scFVs are represented by SEQ ID NOs: 3, 4, 5 and6, respectively.

2. Human MW9 scFv Antibody

For generation of hMW9 scFv, the cDNA for mutant huntingtin exon 1 (HDx)fused to a His tag was expressed in E. coli and purified on nickelcolumns. The purified mutant protein was subjected to SDS-PAGE andtransferred to nitrocellulose membranes. The nitrocellulose membraneswere incubated with a human single-chain fragment variables (scFv) phagelibrary encoding ˜9×10¹⁰ clones. Phage bound to HDx were selected andamplified by infection of susceptible E. coli for 5 rounds. Finally,amplified clones were selected with a recombinant GST-HDX in asolution-based assay. Individual clones were isolated, expressed andrecombinant scFvs were tested for binding to His-HDx in vitro. Positiveclones that bound in vitro were co-expressed with mutant HDx-1 in atissue culture model of Huntington disease and results were evaluatedfor inhibition of cell death. hMW9 scFv expression inhibits aggregationand cell death induced by mutant HDx in this model.

B. Characterization of scFvs

1. Expression Analysis

a. MW1, MW2, MW7 and MW8 scFvs

To test for scFv expression of the MW1, MW2, MW7 and MW8 anti-Httantibodies, 293 cells were transfected with the Flag-tagged scFvs andcell lysates were analyzed by Western blotting and intracellularstaining.

The reading frame for the scFvs were each subcloned into the mammalianplasmid pcDNA3.1 in frame with the Flag epitope for detection purposes(Chiang et al., Pept. Res., 6:62-64 (1993)). Selected clones wereamplified and used to transfect 293 cells that were grown in DMEMsupplemented with 10% heat inactivated bovine serum, 2 mM glutamine, 1mM streptomycin and 100 international units of penicillin. Cells weregrown in 6-well plates to about 70% confluence and transfected with atotal of 2 μg of DNA by using lipofectamine, following themanufacturer's recommendations (Invitrogen). Expression of the scFvswere examined by Western blot analysis of the transfected cell extractsby using an anti-Flag Ab (Sigma).

Full-length proteins for MW1, MW2 and MW7 (FIG. 9A) scFvs were detectedusing anti-Flag Ab.

b. hMW9 scFv

To test for scFv expression of the hMW9 anti-Htt antibodies, control andhMW9 scFvs were expressed by in vitro transcription and translation inthe presence of ³⁵S-methionine. Equal amounts of each were incubatedwith 5 μg of recombinant GST-HDx-1 bound to glutathione beads in abuffer containing mild detergent and glycerol. Following incubations for3 hours at room temperature, the beads were washed 5 times in the bufferwith mild detergent and glycerol. The scFvs that were bound to the beadswere extracted and subjected to SDS-PAGE and autoradiography (FIG. 10).

2. Histological Analysis

For histological examination of 293 cells transfected with Flag-taggedscFvs, transfected cells were fixed in 4% paraformaldehyde for 30minutes at 4° C., permeabilized in 70% methanol at −20° C. for 1 hour,and incubated with anti-Flag Ab (1:1000) for 2 hours. Cells expressingscFvs were detected by a goat anti-mouse Ab conjugated to Alexa 594(Molecular Probes), and examined with a confocal microscope.

Histological examination revealed that the MW1, MW2 and MW7 (FIG. 11)scFvs have a predominantly cytoplasmic distribution.

3. Cell Viability

Because other proteins besides Htt contain polyQ and polyP domains, itwas of interest to test whether expression of the scFvs had an effect oncell viability.

To determine the effects of scFvs on cell viability, human 293 cellswere cotransfected with each scFv and a plasmid encoding enhanced greenfluorescent protein (EGFP; CLONTECH) as a transfection marker to readilydetect which cells were transfected (Nucifora et al., Science,291:2423-2428 (2001)). Viable cells that expressed GFP were counted 4days after transfection by using a fluorescence microscope.

After 4 days of growth, the mean cell counts from at least 30 microscopefields in six wells each revealed no significant differences between thecontrol (112±6), and the scFvs for MW1 (97±3), MW2 (117±4), and MW7(113±5). Accordingly, scFv expression did not affect cell growth orviability.

4. Interaction of Anti-Htt Antibody Fragments with Htt in Living Cells

a. Coimmunoprecipitation

To determine whether the scFvs interact with Htt in living cells,flag-tagged scFvs or flag-tagged IκBα, a control, were coexpressed in293 cells with Htt exon 1 containing either 25 polyQ repeats (PQ25) or103 polyQ repeats (PQ103), fused to EGFP and subjected tocoimmunoprecipitation analysis. The scFvs and IκBα in Triton X-100 cellextracts were precipitated with anti-Flag Ab, and the precipitates weresubjected to SDS-PAGE. The SDS-PAGE gels were analyzed for the presenceof Htt exon 1 by Western blotting using anti-Flag Ab (FIG. 9A) andantibodies specific for the N-terminal 17 amino acids of exon 1 of thehuntingtin protein (FIG. 9B).

Coimmunoprecipitation experiments were performed with 293 cell lysatescotransfected as described above. Briefly, cells were harvested 24 hoursafter transfection and lysed by sonication in buffer A (25 mM Hepes, pH7.4/2.5 mM MgCl₂/50 mM NaCl/1 mM EDTA/1% Triton X-100). After clearingthe lysates by centrifugation at 14,300 rpm (Eppendorf microcentrifuge)for 10 minutes at 4° C., 200 μg of each lysate was incubated at 4° C.with rocking for 2 hours with a 40-μl slurry of anti-Flag Ab coupled toprotein A beads. The beads were then washed five times in buffer A byusing centrifugation at 5,000 rpm, and the complexes were resolved onSDS-PAGE. For Western blotting, rabbit anti-HD1-17 (Mende-Mueller etal., J. Neurosci., 21:1830-1837 (2001)) and anti-Flag (1:1000; Sigma)were used as the primary Abs. Secondary antibodies conjugated tohorseradish peroxidase (HRP) were used to detect the reactive proteinbands by enhanced chemiluminescence (Santa Cruz Biotechnology). TheSDS-PAGE gels were first probed with anti-flag antibodies for thepresence of the scFvs and then stripped and reprobed with an antibodyspecific for the N-terminal 17 amino acids of exon 1 of the huntingtinprotein for the presence of mutant PQ 103 huntingtin about 80 kDa andmutant PQ25 huntingtin about 50 kDa.

As shown in FIG. 9A, similar amounts of each of the MW1, MW2 and MW7scFvs, which migrate with a molecular mass of about 35 kDa and IκBαwhich migrates with a molecular mass of about 43 kDa were precipitatedwith the anti-Flag Ab (FIG. 9A), and mutant Htt exon 1 (PQ103)coimmunoprecipitates with each scFv (FIG. 9B; 86-kDa bands). Similarresults were obtained when Htt exon 1 with a 25-Q stretch (PQ25) wasused for transfection (FIG. 9B; 50 kDa bands). As a negative control, incells expressing Flag-tagged IκBα, IκBα was precipitated from theextract by the anti-Flag Ab (FIG. 9A; 44-kDa band), but Htt was notcoprecipitated (FIG. 9B). The bands below 30 kDa in FIG. 9B likelyrepresented nonspecific staining of the precipitating Ab.

5. Colocalization

To confirm binding of the scFvs to mutant huntingtin protein and tolocalize the sites of interaction within cells, we used confocalmicroscopy to examine 293 cells cotransfected with mutant exon 1 of thehuntingtin protein having 103 Q repeats and fused to EGFP (103-QHtt-EGFP) and each anti-Htt scFv.

For colocalization experiments, the scFvs and 103-Q Htt-EGFP, obtainedfrom the Cure Huntington Disease Initiative Resource Bank (Univ. ofCalifornia, Los Angeles; Steffan et al., Proc. Natl. Acad. Sci. USA,97:6763-6768 (2000)), were cotransfected in 293 cells grown oncoverslips. 24 hours after transfection, cells were fixed, stained, andexamined as described above. Depending on the experiment, 50-70% of thecells expressed EGFP.

Although the MW1, MW2 and MW7 (FIG. 11) scFvs and MW8 scFvs weredistributed throughout the cytoplasm in the absence of Htt, whencotransfected with 103-Q Htt-EGFP, the MW1, MW2 and MW7 (FIG. 13) andMW8 (FIG. 14) scFvs were concentrated in the perinuclear region andcolocalized with 103-Q Htt-EGFP.

6. Effects of Anti-Htt Antibody Fragments on Htt-Induced Cell Death

To evaluate the effect of anti-Htt scFvs on the toxic effects of mutantHtt, we examined by terminal deoxynucleotidyltransferase-mediated dUTPnick end labeling (TUNEL) staining of 293 cells cotransfected with 103-QHtt-EGFP and each scFv.

Two days after cotransfection, 293 cells were fixed as described aboveand washed three times in PBS. The TUNEL reaction consisted of 25 unitsof terminal deoxynucleotidyltransferase and 1 mM dUTP conjugated totetramethylrhodamine (Roche Molecular Biochemicals) in 1× buffer/2.5 mMCoCl₂ in a final volume of 50 μl (according to manufacturer'sinstructions). Coverslips with fixed cells were laid over the reactionmixture and incubated at 37° C. in a humidified incubator. Samples werewashed four times with PBS, mounted on microscope slides, and examinedby confocal microscopy. TUNEL-positive cells that were expressing mutantHtt exon 1 were counted from at least 16 independent microscope fieldswith a ×20 objective lens in four separate experiments. The data wereanalyzed by using EXCEL software to determine the standard deviation andthe P value (t test).

Cells expressing 103-Q Htt-EGFP along with an empty scFv vectordisplayed significant TUNEL staining, and apoptotic bodies were observedstarting about 12 hours after transfection (FIG. 13, control column).TUNEL staining was even more dramatic in the presence of MW1 or MW2 scFvand mutant Htt (FIG. 15). MW1 or MW2 scFv binding to the polyQ domainaccentuated the toxicity of mutant Htt.

Expression of MW7 scFv (FIG. 15) or MW8 scFv (FIG. 16) inhibited thetoxicity of mutant Htt. These experiments were done under the sameconditions as in FIG. 9A, which demonstrated equivalent expression ofMW1, MW2 and MW7 scFvs in the cells.

To quantify the effects of scFv expression on mutant Htt toxicity, wecounted TUNEL+ cells. The increase in mutant Htt-induced TUNEL stainingin the presence of MW1 and −2 scFvs is 38% and 67%, respectively(P<0.05) (FIG. 17). In contrast, the number of TUNEL+ cells in thepresence of MW7 scFv is reduced to 33% of the control (P<0.05) (FIG.15), and the number of TUNEL+ cells in the presence of MW8 scFv isreduced from 72 apoptotic bodies to 26 apoptotic bodies (FIG. 16). Thus,although the anti-polyQ mAbs MW1 and MW2 scFvs accentuate the toxicityof mutant Htt, expression of the anti-polyP mAb MW7 and MW8 scFvsinhibits the toxicity of mutant Htt.

To determine the effects of hMW9 scFv on mutant Htt cell toxicity, human293 cells were cotransfected with the 103-Q Htt-EGFP and hMW9 or ascontrols, an empty plasmid or a control scFv that does not bind to exon1 of the huntingtin protein (HDx-1) by lipofectamine. 103-Q Htt-EGFP,was cotransfected with an empty plasmid (FIG. 12; EGFP-103Q-HDx-1+C),hMW9 (FIG. 12; EGFP-103Q-HDx-1+MW9) or cscFv, a control that does notbind to HDx-1 (FIG. 12; EGFP-103Q-HDx-1+cscFv). Two dayspost-transfection cells were examined by a fluorescent microscope. Cellsremained intact in the presence of hMW9 (middle panel) when compared tothe presence of a control that does not bind to HDx-1 (bottom panel).Without hMW9, mutant Htt results in cell toxicity and the presence ofapoptotic bodies (FIG. 12, top panel). In the presence of hMW9, cellstransfected with mutant Htt are healthy and have less apoptotic bodies(FIG. 12, middle panel).

7. Effects of Anti-Htt Antibody Fragments on Aggregation of Mutant Htt

To evaluate the effects of scFv expression on mutant Htt aggregation in293 cells, Htt aggregation was evaluated biochemically by examining theamount of Htt that precipitated from cell lysates by centrifugation at150,000×g for 30 min.

For aggregation studies, 293 cells were cotransfected with mutant Httexon 1 and an scFv were harvested 48 hours after transfection. Cellswere lysed by sonication in buffer A. Lysates were centrifuged at150,000×g in an SW55 rotor (Beckman Instruments, Fullerton, Calif.) for30 minutes (Nucifora et al., Science, 291:2423-2428 (2001)). Pelletswere dissolved in sample buffer containing 2% SDS, boiled and subjectedto SDS-PAGE, and transferred to nitrocellulose membranes forimmunoblotting analysis. Aggregates were detected with anti-HD1-17polyclonal antibody (Mende-Mueller et al., J. Neurosci., 21:1830-1837(2001)).

The pellets contained aggregated Htt (or Htt that is bound to largestructures) that can be solubilized by SDS treatment (FIG. 18A, 80-kDabands), as well as Htt that remained insoluble after boiling in SDS andcannot enter the gel (FIG. 18A, top of gel). Both such species ofpelleted Htt were detected in extracts of cells transfected with mutantHtt exon 1 alone (FIG. 18A). Aggregation increased when mutant Htt exon1 was coexpressed with MW1 scFv or MW2 scFv. Very little aggregated Httwas found in the presence of MW7, MW8 or hMW9 scFv. Scanning the bandsat the top of the gel in FIG. 18 yielded values in arbitrary units of68.8 for MW1, 54.3 for MW2, 0.2 for MW7, and 48.8 for no scFv.

Accordingly, coexpression of MW7 scFv interfered with aggregation ofmutant Htt exon 1, and there was a qualitative correlation between theeffects of the scFvs on Htt aggregation and toxicity. The expression ofMW7 did not cause a depletion in the level of soluble Htt (FIG. 18B).

Coexpression of hMW9 also interfered with aggregation of mutant Htt exon1.

1. An isolated monoclonal antibody that specifically binds an epitopewithin a polyproline region of the huntingtin protein comprising greaterthan 5 consecutive proline residues and wherein the antibody is capableof inhibiting aggregation of the huntingtin protein.
 2. The monoclonalantibody of claim 1, in association with a therapeutically acceptablecarrier.
 3. A method for treatment of Huntington's disease, comprisingadministering to a patient an effective amount of a monoclonal antibodyof claim
 1. 4. The method of claim 3 wherein said monoclonal antibody isa single-chain variant fragment encoded by the nucleotide sequence ofSEQ ID NO:
 5. 5. The method of claim 3 wherein the patient is amammalian patient.
 6. The method of claim 5 wherein the mammalianpatient is human.
 7. The method of claim 6 wherein the antibody isdelivered intracranially.
 8. The method of claim 7 wherein the antibodyis injected directly into brain tissue.
 9. The method of claim 7 whereinthe antibody is injected into the cerebrospinal fluid.
 10. A method fortreatment of Huntington's disease, comprising expression of a monoclonalantibody of claim 1 in cells expressing mutant huntingtin protein. 11.The method of claim 10 wherein said monoclonal antibody is asingle-chain variant fragment encoded by the nucleotide sequence of SEQID NO: 5.